100,000 Americans perish each year due to untreatable bacterial infections. The societal benefits of new antibiotic compounds that are effective against numerous multiple drug resistant pathogens would be significant. The best possible source for new antibiotic structures with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. This research proposal advances the science of metagenomics, the cloning of DNA from entire microbial communities, to discover novel antibiotics and identify the best lead candidates for clinical development. During Phase I research scientists at Lucigen Corporation and Auburn University united four key technological breakthroughs that together resulted in the next generation metagenomic library. This library combined 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms; 2) a new broad host range shuttle vector for enhanced expression of cloned DNAs; 3) a random shear cloning method to produce very large insert sizes (>100 kb); and 4) a rapid and improved screening method to identify antibiotic-producing clones within a metagenomic library. The library produced in Phase I was screened against a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the identification of 28 metagenomic clones that produce anti- MRSA compounds. 12 of these anti-MRSA clones were analyzed by sequencing and found to have very large insert sizes (average 113.5 kb) and novel genetic diversity not encountered before. Moreover, one of the clones was found to produce a novel chemical metabolite. These results are 10-100 fold more efficient than previous efforts. In Phase II a large metagenomic library will be constructed and extensively screened for antimicrobial activity against four multiple drug resistant pathogens. We expect to uncover hundreds of novel chemical entities using this approach, and lead candidates with high potency against multiple bacterial pathogens will be evaluated for efficacy using a novel in vivo assay of MRSA. These technologies represent an important advancement for the science of antibiotic discovery. Furthermore, the libraries produced from this research are a valuable genomic resource that may be screened for other bioactive compounds (e.g., anticancer, antifungal or antiviral activities).